Abstract Q fever is a worldwide zoonotic disease that is caused by the obligate intracellular Gram-negative bacterium, Coxiella burnetii. Human Q fever can develop into a severe chronic, potentially fatal disease. Although this organism previously weaponized and currently classified as a category B select agent, no vaccine is commercially available for the prevention of human Q fever in the US. Thus, the creation of a safe and effective vaccine for preventing Q fever remains an important goal for public health and national biosecurity. To achieve this goal, this revised R21 application aims to explore the feasibility of designing a multivalent mimetic peptide Q fever vaccine targeting novel neutralization-sensitive epitopes of C. burnetii T cell-independent antigens. Despite C. burnetii being an obligate intracellular bacterial pathogen, our recent work demonstrated that formalin-inactivated Nine Mile phase I whole cell vaccine (PIV)-induced protection is B-cell dependent, requiring IgM and IgG, and that PIV-specific T cell-independent IgM plays a critical role. Thus, antigens that activate B cells to produce PIV-specific IgM are expected to be promising vaccine candidates. Interestingly, our previous studies demonstrated that a peptide mimic of a C. burnetii phase I lipopolysaccharides protective epitope (m1E41920) conjugated to keyhole limpet haemocyanin (m1E41920-KLH) conferred significant protection against C. burnetii infection. This finding supports the utility of m1E41920 as a vaccine candidate to prevent human Q fever. However, m1E41920-KLH did not confer the same level of protection as the PIV. Thus, the overall objective of this application is to define a multivalent mimotope Q fever vaccine that confers the equal level of protection as the PIV. To achieve this objective, we propose one specific aim to test the central hypothesis that a humoral response to multiple neutralization-sensitive epitopes of C. burnetii T cell- independent antigens can confer the same level of protection as the PIV. We will identify additional protective mimetic peptides of T cell-independent antigens to enhance the protection conferred by this monovalent m1E41920-based vaccine. Upon the completion of this project, we expect to define the first multivalent mimotope Q fever vaccine that targets novel neutralization-sensitive epitopes of T cell-independent antigens.